Pressures caused by daily oscillations in the environment have favored the evolution of organisms that have adopted 24‐hour rhythmic cycles, or circadian rhythms. Daily oscillations in mammalian physiology are generated through an intrinsic molecular‐clock mechanism. An unresolved phenomenon in the field of circadian rhythms is the underlying mechanism(s) that allow the core molecular‐clock proteins (Bmal1/Clock common across all cell types) to transcriptionally target genes in a tissue‐specific fashion. The goal of this project was to use a targeted approach in skeletal muscle to identify mechanisms in which the molecular‐clock regulates the transcription of skeletal muscle specific genes. We focused our study on the molecular regulation of the muscle specific z‐line gene Titin‐cap (T‐cap). We choose this gene because it is highly circadian in skeletal muscle, it is known to be regulated downstream of the myogenic regulatory factor MyoD1 in skeletal muscle and its expression is significantly down‐regulated in the muscle of Bmal1 KO mice. Utilizing T‐cap promoter‐reporter contstructs in C2C12 myotubes, we demonstrate that the T‐cap promoter contains essential elements required for circadian oscillation in vitro (absence of environmental time cues) suggestive of a direct clock‐controlled gene. We performed a series of transfection experiments testing T‐cap promoter‐reporter constructs in C2C12 myotubes, and demonstrated that T‐cap is transactivated by overexpression of clock factors BMAL1:CLOCK, as well as MyoD1. We demonstrate that the T‐cap promoter contains essential elements required for circadian oscillation in vitro (absence of environmental time cues) suggestive of a direct clock‐controlled gene. Overexpression of MyoD1 was sufficient to enhance the amplitude of the T‐cap promoter‐reporter rhythm without affecting the circadian phase or period length. Interestingly, co‐transfection studies with mutant forms of Bmal1 or MyoD1, showed that BMAL1:CLOCK and MyoD1 work in a synergistic fashion and require each other to promote the circadian expression of T‐cap in C2C12 myotubes. Deletion constructs and mutagenesis studies followed by ChIP assays demonstrated that both BMAL1 and MyoD1 were bound to the proximal promoter of T‐cap, and we identified a tandem E‐box element required for T‐cap circadian expression. These results demonstrate that the molecular‐clock factors, BMAL1 and CLOCK, work with MyoD1, a tissue‐specific transcription factor, to promote the circadian expression of T‐cap in skeletal muscle. On going studies are aimed at elucidating the molecular basis underlying the synergist interaction observed between the molecular‐clock and MyoD1.Support or Funding InformationNIH RO1: AR066082